The studies outlined in this proposal are a continuation of our observations on the inhibition of bacterial growth in vitro and in vivo by beta-chloro-D-alanine. The rationale for the inception of these experiments was based upon the reported irreversible inhibition of pyridoxal phosphate enzymes for L-amino acids by beta-chloro-L-amino acids. Our thesis - that beta-chloro-D-alanine might inactivate irreversibly the bacterial enzymes that catalyze the formation of the D-alanine necessary for cell wall synthesis - has been supported by the preliminary experimental data obtained to date. A detailed study into the mechanism of inactivation of both alanine racemase and D-glutamate-D-alanine transaminase by beta-chloro-D-alanine will be undertaken; the possibility that beta-chloro-D-alanine is incorporated into cell wall precursors will also be investigated. Comparative studies on the extent of interaction of both isomers of beta-chloroalanine with cellular intermediates and enzymes will be initiated. Other beta-chloroamino acids, such as the isomers of beta-chloroglutamic acid, will also be investigated for their anti-bacterial properties. The intracellular free alanine of E. coli and B. subtilis was found to be predominantly the D-isomer in untreated cells and the L-isomer in cells that had been treated with beta-chloro-D-alanine. These observations will be extended by similar studies with other microorganisms. The influence of the L-alanine dehydrogenase, which is inducible by D-alanine, on the intracellular alanine pools will be studied. Preliminary studies, which indicated the effectiveness of beta-chloro-D-alanine on the inhibition of bacterial growth in mice, will be extended to include a wide variety of microorganisms - those that are pathogenic and some strains that are resistant to penicillin.